Notch3 is an intriguing plasma membrane-bound transcription factor that has recently been implicated in lung cancer. To define the Notch3 regulatory pathway in lung cancer, we propose to use a mass spectrometry-based proteomics approach to identify and characterize proteins that interact with Notch3 in lung tumor cells. We will use either (a) mono-specific antibodies or (b) Tandem Affinity Purification (TAP) protocols to recover selected Notch3 protein complexes from cultured human lung cells. The purified protein complexes will be identified using a high sensitivity mass spectrometry approach developed in my sponsor's laboratory. This approach couples microcapillary chromatography with mass spectrometry and genome database analysis to directly identify components of purified protein complexes. From a bioinformatics analysis, the identified proteins thought to be relevant to lung cancer development or Notch3 function will be given priority for further examination. Notch3 interactions with these candidate proteins will be validated using a variety of biochemical approaches. This list of validated Notch3 protein interactions will be used to model the regulatory protein network controlling Notch3's regulation of transcription events. A subset of validated proteins with suspected roles in Notch3 transcription will be selected for Notch3 functional studies. We anticipate that this study will help define some novel molecular events that contribute to lung cancer biology. Furthermore, the development of methodology in this work should be applicable to other target proteins and disease states for the discovery of novel protein-protein interaction for diagnostic or therapeutic applications.